Molecular cloning and heterologous expression of novel glucosyltransferases from tobacco cultured cells that have broad substrate specificity and are induced by salicylic acid and auxin.

Scopoletin is one of the phytoalexins in tobacco. Cells of the T-13 cell line (Nicotiana tabacum L. Bright Yellow) accumulate a large amount of scopoletin, also known as 7-hydroxy-6-methoxycoumarin, as a glucoconjugate, scopolin, in vacuoles. We report here the molecular cloning of glucosyltransferases that can catalyze the glucosylation of many kinds of secondary metabolites including scopoletin. Two cDNAs encoding glucosyltransferase (NtGT1a and NtGT1b) were isolated from a cDNA library derived from the tobacco T-13 cell line by screening with heterologous cDNAs as a probe. The deduced amino-acid sequences of NtGT1a and NtGT1b exhibited 92% identity with each other, approximately 20-50% identities with other reported glucosyltransferases. Heterologous expression of these genes in Escherichia coli showed that the recombinant enzymes had glucosylation activity against both flavonoids and coumarins. They also strongly reacted with 2-naphthol as a substrate. These recombinant enzymes can utilize UDP-glucose as the sugar donor, but they can also utilize UDP-xylose as a weak donor. RNA blot analysis showed that these genes are induced by salicylic acid and auxin, but the time course of the expression was different. This result is similar to the changes in scopoletin glucosylation activity in these tobacco cells after addition of these plant growth regulators. These results might suggest that one of the roles of the products of these genes is scopoletin glucosylation, in response to salicylic acid and/or auxin, together with the other glucosyltransferases in tobacco cells.

Increases of secondary metabolite production in various plant cell cultures by co-cultivation with cork.

Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors. In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones. The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species. Woody tissues of Japanese cypress had a very similar effect to that of cork. Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues.

Plant hormone regulation on scopoletin metabolism from culture medium into tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has an ability for production of scopoletin. In this cell culture, scopoletin is taken up from culture medium and accumulated in vacuoles after conversion to scopolin when cells are treated with 2,4-dichlorophenoxyacetic acid (2,4-D) (Taguchi et al. (2000)). To clarify the effect of 2,4-D on tobacco cells, its interaction with several other plant hormones was investigated. Other auxins also stimulated the uptake in the same manner as 2,4-D did, although higher concentrations were required than that of 2,4-D. When p-chlorophenoxyisobutyric acid (PCIB), an antiauxin, was added to the cell culture before 2,4-D, it inhibited 2,4-D-stimulated scopoletin uptake. This result suggests that the stimulation of scopoletin uptake was one of the auxin effects on tobacco cells. Among other classes of plant hormones that were tested, only salicylic acid stimulated the uptake. When these hormones were added to the cell cultures before 2,4-D, methyl jasmonate and kinetin reduced scopoletin uptake. These results suggest that this scopoletin uptake by tobacco cells is regulated by the interaction between different plant hormones.

Purification and characterization of UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase, with broad substrate specificity from tobacco cultured cells.

The enzyme UDP-glucose: hydroxycoumarin 7-O-glucosyltransferase (CGTase), which catalyzes the formation of scopolin from scopoletin, was purified approximately 1200-fold from a culture of 2,4-D-treated tobacco cells (Nicotiana tabacum L. cv. Bright Yellow T-13) with a yield of 7%. Purification to apparent homogeneity, as judged by SDS-PAGE, was achieved by sequential anion-exchange chromatography, hydroxyapatite chromatography, gel filtration, a second round of anion-exchange chromatography, and affinity chromatography on UDP-glucuronic acid agarose. The purified enzyme had a pH optimum of 7.5, an isoelectric point (pI) of 5.0, and a molecular mass of 49 kDa. The enzyme did not require metal cofactors for activity. Its activity was inhibited by Zn(2+), Co(2+) and Cu(2+) ions, as well as by SH-blocking reagents. The K(m) values for UDP-glucose, scopoletin and esculetin were 43, 150 and 25 µM, respectively. A study of the initial rate of the reaction suggested that the reaction proceeded via a sequential mechanism. The purified enzyme preferred hydroxycoumarins as substrates but also exhibited significant activity with flavonoids. A database search using the amino terminus amino acid sequence of CGTase revealed strong homology to the amino acid sequences of other glucosyltransferases in plants.

Scopoletin uptake from culture medium and accumulation in the vacuoles after conversion to scopolin in 2,4-D-treated tobacco cells.

Tobacco (Nicotiana tabacum L. Bright Yellow) T-13 cell line has the ability to produce scopoletin endogenously and release some of it into the culture medium. We investigated the mechanism of scopoletin uptake following treatment of a tobacco culture with 2,4-dichlorophenoxyacetic acid (2,4-D). Addition of [14C]-labeled scopoletin showed that scopoletin was taken up by 2,4-D-treated cells and converted to scopolin, a 7-O-glucoside of scopoletin. This uptake of scopoletin began 6 h after 2,4-D addition to the cells. Experiments using several inhibitors showed that this uptake was energy-dependent. The phenomenon of 2,4-D-stimulated uptake was observed only for 7-hydroxycoumarins, such as scopoletin, umbelliferone and esculetin. To further investigate the site for scopoletin accumulation, we separated the vacuoles from T-13 cells and quantified the coumarin contents in this fraction. Most of the scopoletin in the vacuoles was present as glucoconjugate, scopolin. Moreover, glucosylation activity was absent from isolated vacuoles and, therefore, is likely to be located in the cytosol. Therefore, we can state that 2,4-D treatment of tobacco cells stimulated scopoletin uptake. The scopoletin was converted into scopolin in the cytoplasm, and then transferred into the vacuoles.

Cloning and characterization of mycovirus double-stranded RNA from the plant pathogenic fungus, Fusarium solani f. sp. robiniae.

A mycovirus (named FusoV) from the phytopathogenic fungus, Fusarium solani f. sp. robiniae SUF704, has two kinds of double-stranded (ds) RNA genomes, designated M1 and M2. The cDNAs were constructed from FusoV genomic dsRNAs. The sequences of M1 and M2 cDNAs comprised 1645 and 1445bp, respectively. Sequence analysis showed that each dsRNA had a single long open reading frame (ORF) on only one of the strands. M1 ORF encodes a 519-amino acid residue polypeptide with a predicted molecular mass of 60 kDa. RNA-dependent RNA polymerase-conserved motifs were identified in the predicted amino acid sequence, and the polymerase synthesized dsRNA in vitro. The M2 ORF encodes a polypeptide of 413 amino acid residues with a predicted molecular mass of 44 kDa. The predicted amino acid sequence contained the sequence corresponding to those found in the purified 44-kDa capsid protein of FusoV.

[Phase III controlled studies of bestatin in malignant tumors of the skin--results of treatment of squamous cell carcinoma and genital Paget's disease].

We have performed a co-operative controlled clinical study of Bestatin in the treatment of malignant tumors of the skin at 18 research institutions in Japan. Twenty-seven Bestatin-treated cases of stage I and II squamous cell carcinoma and 24 control cases were followed up for a maximum of 46 months, to compare disease-free and survival rates in the 2 groups. There was no significant difference in either rate between the groups, though each rate was slightly higher for the Bestatin-treated group. Eight Bestatin-treated cases of genital Paget's disease and 8 control cases were similarly followed up. The survival rate was slightly higher for the Bestatin-treated group, but each group consisted of too small a number of cases to demonstrate the utility of Bestatin. The data obtained up to now only suggest the future potentiality of Bestatin treatment for these types of malignancy. For this reason, the data from phase III studies of Bestatin, including previously reported findings on malignant melanoma, proved Bestatin to be useful in treating stage Ib and II malignant melanoma, but were only suggestive of its potential indication in the treatment of other malignant tumors of the skin. Although Bestatin was administered for long periods (a mean of 21.5 months), only mild gastrointestinal disturbances were observed as adverse reactions to the drug in 4.5% of cases.

[Phase III study of bestatin in patients with malignant skin tumors. (1) Malignant melanoma].

As a co-operative study involving 18 medical research institutions, we have made a randomized, controlled study (phase III study) of Bestatin in the treatment of malignant melanoma (stage Ib, II) over the last 4 years. The results were as follows; Bestatin significantly prolonged disease-free intervals by 9-20 month. Bestatin significantly prolonged survival periods by 10-16 months. Side effects of Bestatin, chiefly comprising of reversible gastro-intestinal disturbances, were observed in 7.1% of Bestatin-treated cases. These findings suggest that Bestatin is a very useful agent for treating for stage Ib and II malignant melanomas.